Organic-arsenic compounds

ABSTRACT

Novel organic arsenic compounds are described as cytotoxic agents with potent anti-tumor activity against cancer cells and particularly, with regard to human leukemic cells and breast cancer cells.

PRIORITY OF INVENTION

This application is a divisional of application Ser. No. 09/338,680,filed Jun. 23, 1999, now U.S. Pat. No. 6,191,123 which claims benefit ofpriority to provisional application Ser. No. 60/125,337, filed Mar. 19,1999, which applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel organic arsenic compounds for treatingtumor cells and which are particularly effective for inducing apoptosisin leukemia and breast tumor cells.

BACKGROUND OF THE INVENTION

Cancer is a major disease that continues as one of the leading causes ofdeath at any age. In the United States alone, it is anticipated thatmore than a half a million Americans will die of cancer in 1999.Currently, radiotherapy and chemotherapy are two important methods usedin the treatment of cancer.

Considerable efforts are underway to develop new chemotherapeutic agentsfor more potent and specific anti-cancer therapy, presenting effectiveand efficient cytotoxicity against tumor cells, with minimalinterference with normal cell function. Accordingly, there is an urgentneed for the development and analysis of novel, effective anti-canceragents.

SUMMARY OF THE INVENTION

New organic arsenic acid substituted cytotoxic agents with potentanti-tumor activity against cancer cells have been synthesized andexamined for their effect on human leukemic cells and breast cancercells. The compounds were found to exhibit potent cytotoxic activity,particularly against human breast cancer and leukemic cell lines,including primary leukemia cells, at micromolar concentrations.

Accordingly, the present invention includes novel compounds andcompositions having potent cytotoxic activity. The present inventionalso includes methods for treating tumors by administering to a subjectan anti-tumor effective amount of a compound of the invention.Compositions of the invention contain an effective or inhibitory amountof a organic arsenic acid substituted compound. The compounds of theinvention include those having the following formula:

where R is

R¹ is selected from a group consisting of H, NR³R⁴, SR³ and OR³, inwhich R³ and R⁴ are each independently hydrogen or a C₁-C₄ alkyl group.X is N or C. R² is selected from the group consisting of H, NR³R⁴, SR³,OR³, and a group capable of bonding with X, when X is C, to form a fusedaromatic or 5- or 6-membered heteroaromatic ring, or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are photographs showing cells incubated with 2 μM WHI-P381for 24 hours, fixed in 2% paraformaldehyde, permeabilized, andvisualized for DNA fragmentation (apoptosis assay). Shown are NALM-6cells untreated (1A) and treated with WHI-P381 (1B); MOLT-3 cellsuntreated (1C) and treated with WHI-P381 (1D).

FIGS. 2A-2D are cell survival graphs demonstrating time anddose-dependent cytotoxic activity of WH-P381 compared with arsenictrioxide against leukemic NALM-6 cells after 1 (FIG. 2A), 2 (FIG. 1B), 3(Fig. 1C), and 4 (FIG. 1D) days of treatment.

FIGS. 3A-3F are cell survival graphs demonstrating dose-dependentcytotoxitity of organic arsenic compounds on leukemia ALL (FIGS. 3A-3B),NALM-6 (FIGS. 3C-3D), and MOLT-3 (FIGS. 3E-3D) cells.

FIGS. 4A-4E are cell survival graphs demonstrating the cytotoxicactivity of organic arsenic acid substituted compounds against primaryleukemic cells. Shown are % survival of cells from five patients (FIGS.4A-4E) treated with WHI-P273, WHI-P370, WFH-P371, WHI-P380, or WHI-P381.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel organic arsenic acid substitutedcompounds having potent activity as cytotoxic agents. The compounds ofthe invention are useful agents in treating tumor cells, for example,against leukemia and breast tumor cells. The organic arsenic acidsubstituted compounds of the invention are effective in inducingapoptosis in leukemia and breast tumor cells.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. As a preferred embodiment, chains of 1 to 4 carbon atomsare included, for example methyl, ethyl, propyl, isopropyl, butyl,secondary butyl, t-butyl, and the like.

As used herein “halogen” or “halo” substituent includes fluoro, chloro,bromo, and iodo.

As used herein, “fused aromatic ring” includes an unsubstituted orsubstituted benzene or napthalene ring.

As used herein, “fused heteroaromatic ring” includes a 5- or 6-memberedheterocyclic ring having at least one heteroatom selected from nitrogen,oxygen and sulfir.

As used herein, “pharmaceutically acceptable salt thereof” includes anacid addition salt or a base salt.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with a compound of the invention, allowsthe compound to retain biological activity, such as the ability toinduce apoptosis of leukemia or breast tumor cells, and is non-reactivewith the subject's immune system. Examples include, but are not limitedto, any of the standard pharmaceutical carriers such as a phosphatebuffered saline solution, water, emulsions such as oil/water emulsions,and various types of wetting agents. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., MackPublishing Co., Easton, Pa.).

Compounds of the Invention

The novel organic arsenic acid substituted compounds of the inventionhave the general structure represented by the following formula I:

where R is

R¹ is H, NR³R⁴, SR³, OR³, in which R³ and R⁴ are each independentlyhydrogen or a C₁-C₄ alkyl group. Preferably, R³ is hydrogen or methyl,and R⁴ is hydrogen. X is N or C.

R² is H, NR³R⁴, SR³, OR³, or a group capable of bonding with X, when Xis C, to form a fused aromatic or 5- or 6-membered heteroaromatic ring,or a pharmaceutically acceptable salt thereof.

In one embodiment, the fused aromatic ring is preferably a benzene orMapthalene ring, which ring is unsubstituted or substituted by one ormore groups selected from halo, hydroxy, mercapto, alkyl of 1-4 carbonatoms, alkoxy of 1-4 carbon atoms, thioalkyl of 1-4 carbon atoms,hydroxyalkyl of 1-4 carbon atoms, NR³R⁴, nitro, cyano, CF₃, COOH, SO₃H,SO₂NR³R⁴ in which R³ and R⁴ are as defined above, and SO₂F. Morepreferably, the fused aromatic ring is a benzene ring unsubstituted orsubstituted by one or more groups selected from halo, hydroxy, C₁-C₄alkoxy or trifluoromethyl. The benzene ring is most preferably3,4-dimethoxy benzene.

In an alternate embodiment, the fused heteroaromatic ring is preferablya 5- or 6-membered unsaturated heterocyclic ring having at least oneheteroatom selected from nitrogen, oxygen and sulfur. More preferably,the fused heteroaromatic ring is a 5-membered ring having at least onenitrogen atom. Most preferably, the 5-membered ring having at least onenitrogen atom, is imidazole.

The compounds of the invention are capable of forming bothpharmaceutically acceptable acid addition and/or base salts. Base saltsare formed with metals or amines, such as alkali and alkaline earthmetals or organic amines. Examples of metals used as cations are sodium,potassium, magnesium, calcium, and the like. Also included are heavymetal salts such as for example silver, zinc, cobalt, and cerium.Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamnene,N-methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts are formed with organicand inorganic acids. Examples of suitable acids for salt formation arehydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, gluconic, fumaric, succinic, ascorbic, maleic,methanesulfonic, and the like. The salts are prepared by contacting thefree base form with a sufficient amount of the desired acid to produceeither a mono or di, etc. salt in the conventional manner. The free baseforms may be regenerated by treating the salt form with a base. Forexample, dilute solutions of aqueous base may be utilized. Diluteaqueous sodium hydroxide, potassium carbonate, ammonia, and sodiumbicarbonate solutions are suitable for this purpose. The free base formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but the salts areotherwise equivalent to their respective free base forms for thepurposes of the invention.

The organic arsenic acid substituted compounds of the present inventioncan be prepared by the condensation of, for example, quinazoline,pyrimidine, triazine or purine derivatives and a organic arsenic acidderivative as shown in Scheme 1. R, R¹ and R² in Scheme 1 represent thegroups previously defined. The reactants, which are either commerciallyavailable or prepared by known methods, are heated to reflux in anappropriate solvent for a period of time up to 24 hours. An excessamount of triethylamine is added and the solvent evaporated to affordthe crude product which is purified by recrystallization.

Cytotoxic Compounds

As shown in the examples below, the organic arsenic acid substitutedcompounds of the invention are effective cytotoxic agents, useful, forexample, against tumor cells such as leukemic and breast cancer cells.In the methods of the invention, the cytotoxic effect of these compoundsis achieved by contacting the target cell with micromolar amounts of theinhibitory compound.

Particularly useful compounds having potent cytotoxic effects againstleukemia cells include:

4-[(6′,7′-dimethoxyquinazoline-4′)-aminophenylazo]organic arsenic acid(WHI-P273);

2-methylthio-4-[(4′-aminophenylazo)-organic arsenic acid]pyrimidine(WHI-P370);

6-[(4′-aminophenylazo)-organic arsenic acid]-purine (WHI-P371);

2,6-diamino-4[(4′-aminophenylazo)-organic arsenic acid]-1,3,5-triazine(WHI-P374);

2,6-dimethoxy-4[(4′-aminophenylazo)-organic arsenic acid]-1,3,5-triazine(WHI-P376);

4-(2′-organic arsenic acid)-amino-6,7-dimethoxyquinazoline (WHI-P378);

2-methylthio-4-(4′-organic arsenic acid)-aminopyrimidine (WHI-P380);and

2-methylthio-4-(2′-organic arsenic acid)-aminopyriridine (WHI-P381).

Of these compounds, WHI-P273, WHI-P370, WHI-P371, WHI-P380 and WHI-P381are particularly potent for inducing apoptosis in leukemia cells. Mostuseful are WHI-P380 and WHI-P381.

Particularly useful compounds having potent cytotoxic effects againstbreast cancer cells include WHI-P370, WHI-P374, WHI-P376 and WHI-P381.Of these compounds, WHI-P374, WHI-P376 and WHI-P381 are moreparticularly potent for inducing apoptosis in breast cancer cells.

WHI-P381 is a particularly useful compound having a potent cytotoxiceffect against both leukemia and breast tumor cells.

Tumor Treatment

For purposes of this invention, a method of tumor treatment includesadministering to a subject a compound of the invention in order toachieve an inhibition of tumor cell growth, a killing of tumor cells,reduction of tumor size, induction of cellular apoptosis, and/orincreased patient survival time.

The cytotoxic compounds of the invention are suitable for use inmammals. As used herein, “mammals” means any class of higher vertebratesthat nourish their young with milk secreted by mammary glands,including, for example, humans, rabbits, and monkeys.

Apoptosis

Apoptosis, or programmed cellular death, is an active process requiringnew protein synthesis. Typically, the process requires ATP, involves newRNA and protein synthesis, and culminates in the activation ofendogenous endonucleases that degrade the DNA of the cell, therebydestroying the genetic template required for cellular homostasis.Apoptosis is observed in controlled deletion of cells duringmetamorphosis, differentiation, and general cell turnover and appearsnormally to be regulated by receptor-coupled events. For these reasons,apoptosis has been called “programmed cell death” or “cell suicide.”While every cell likely has the genetic program to commit suicide, it isusually suppressed. Under normal circumstances, only those cells nolonger required by the organism activate this self-destruction program.

Apoptotic cell death is characterized by plasma membrane blebbing, cellvolume loss, nuclear condensation, and endonucleolytic degradation ofDNA at nucleosome intervals. Loss of plasma membrane integrity is arelatively late event in apoptosis, unlike the form of cell death termednecrosis, which can be caused by hypoxia and exposure to certain toxinsand which is typically characterized early-on by increased membranepermeability and cell rupture.

Administration Methods

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host,including a human patient in a variety of forms adapted to the chosenroute of administration. The compounds are preferably administered incombination with a pharmaceutically acceptable carrier, and may becombined with specific delivery agents, including targeting antibodiesand/or cytokines.

The compounds can be administered orally, parentally (includingsubcutaneous injection, intravenous, intramuscular, intrasternal orinfusion techniques), by inhalation spray, topically, by absorptionthrough a mucous membrane, or rectally, in dosage unit formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants or vehicles. Pharmaceutical compositions of the invention canbe in the form of suspensions or tablets suitable for oraladministration, nasal sprays, creams, sterile injectable preparations,such as sterile injectable aqueous or oleagenous suspensions orsuppositories.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Preferred administration routes include orally, parenterally, as well asintravenous, intramuscular or subcutaneous routes.

More preferably, the compounds of the present invention are administeredparenterally, i.e., intravenously or intraperitoneally, by infusion orinjection. In one embodiment of the invention, the compounds may beadministered directly to a tumor by tumor injection; or by systemicdelivery by intravenous injection.

Solutions or suspensions of the compounds can be prepared in water,isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions or dispersions or sterile powderscomprising an active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.In all cases, the ultimate dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols and the like, vegetableoils, nontoxic glyceryl esters, and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption—for example, aluminummonosterate hydrogels and gelatin.

Sterile injectable solutions are prepared by incorporating theconjugates in the required amount in the appropriate solvent withvarious other ingredients as enumerated above and, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying techniques, which yielda powder of the active ingredient plus any additional desired ingredientpresent in the previously sterile-filtered solutions.

Useful Dose

When used in vivo to kill tumor cells, the administered dose is thateffective to have the desired effect, e.g., sufficient to reduce oreliminate tumors. Appropriate amounts can be determined by those skilledin the art, extrapolating using known methods and relationships, fromthe in vitro data provided in the Examples.

In general, the dose of the novel organic arsenic acid substitutedcompounds effective to achieve tumor cell apoptosis, reduction intumors, and increased survival time, is that which administersmicromolar amounts of the compound to the cells, preferably 100micromolar or greater. The required dose is lessened by conjugation ofthe compound to a targeting moiety, for example, to preferably 100nanomolar or greater concentrations.

The effective dose to be administered will vary with conditions specificto each patient. In general, factors such as the disease burden, tumorlocation (exposed or remote), host age, metabolism, sickness, priorexposure to drugs, and the like contribute to the expected effectivenessof a drug. One skilled in the art will use standard procedures andpatient analysis to calculate the appropriate dose, extrapolating fromthe data provided in the Examples.

In general, a dose which delivers about 1-100 mg/kg body weight isexpected to be effective, although more or less may be useful.

In addition, the compositions of the invention may be administered incombination with other anti-tumor therapies. In such combinationtherapy, the administered dose of the organic arsenic acid substitutedcompounds would be less than for single drug therapy.

Conjugation to a Targeting Moiety

The compound of the invention can be targeted for specific delivery tothe cells to be treated by conjugation of the compounds to a targetingmoiety. Targeting moiety useful for conjugation to the compounds of theinvention include antibodies, cytokines, and receptor ligands expressedon the cells to be treated.

The term “conjugate” means a complex formed with two or more compounds.

The phrase “targeting moiety” means a compound which serves to deliverthe compound of the invention to a specific site for the desiredactivity. Targeting moieties include, for example, molecules whichspecifically bind molecules present on a cell surface. Such targetingmoieties useful in the invention include anti-cell surface antigenantibodies. Cytokines, including interleukins, factors such as epidermalgrowth factor (EGF), and the like, are also specific targeting moietiesknown to bind cells expressing high levels of their receptors.

Particularly useful targeting moieties for targeting the compounds ofthe invention to cells for therapeutic activity include those ligandsthat bind antigens or receptors present on the tumor cells to betreated. For example, antigens present on B-lineage cancer cells, suchas CD19, can be targeted with anti-CD19 antibodies such as B43. Antibodyfragments, including single chain fragments, can also be used. IL4 canalso be used to target B-cells. Cancer cells expressing EGF or IGFreceptors can be targeted with the binding ligand. Other suchligand-receptor binding pairs are known in the scientific literature forspecific cancers. Methods for producing conjugates of the compounds ofthe invention and the targeting moieties are known.

EXAMPLES

The invention may be farther clarified by reference to the followingExamples, which serve to exemplify some of the embodiments, and not tolimit the invention in any way.

Example 1 Synthesis of Substituted Organic Arsenic Compounds

All chemicals were purchased from the Aldrich Chemical Company,Milwaukee, Wis., and were used directly for synthesis. Anhydroussolvents such as acetonitrile, methanol, ethanol, ethyl acetate,tetrahydrofuran, chloroform, and methylene chloride were obtained fromAldrich as sure seal bottles under nitrogen and were transferred toreaction vessels by cannulation. All reactions were carried out under anitrogen atmosphere.

The organic arsenic acid substituted compounds of the present inventionwere prepared by the condensation of quinazoline, pyrimidine, triazineor purine and organic arsenic acid according to the procedure shown inScheme 1.

where R is

R¹ is H, NR³R⁴, SR³, OR³, in which R³ and R⁴ are each independentlyhydrogen or a C₁-C₄ alkyl group. Preferably, R³is hydrogen or methyl,and R⁴ is hydrogen. X is N or C.

R² is H, NR³R⁴, SR³, OR³, or a group capable of bonding with X, when Xis C, to form a fused aromatic or 5- or 6-membered heteroaromatic ring,or a pharmaceutically acceptable salt thereof.

The reactants, which are either commercially available or prepared byknown methods, were chosen as appropriate for the synthesis of thecompound desired, and heated to reflux in an appropriate solvent for aperiod of time up to 24 hours. An excess amount of triethylamine wasadded and the solvent evaporated to afford the crude product which waspurified by recrystallization.

Specifically, to prepare WHI-378, a mixture of 4-Cl-quinazoline (2mmols) and o-Arsanilic acid (3 mmols) in EtOH (20 ml) was heated toreflux. After refluxing for 24 hours, an excess amount of Et₃N wasadded, and the solvent was concentrated to give the crude product(WHI-378) which was recrystallized from DMF.

Additional compounds, including those shown below in Table 2, weresynthesized using this method, and with appropriate reactants.

Example 2 Characterization of Substituted Quinazoline Derivatives

The following organic arsenic acid substituted compounds weresynthesized as described above and characterized. Each structure isshown below in Table 1. The identifying analytical test results for eachcompound are also shown below. Proton and carbon Nuclear MagneticResonance (¹H and ¹³C NMR) spectra were recorded on a Mercury 2000Varian spectrometer operating at 300 MHz and 75 MHz, respectively, usingan automatic broad band probe. Unless otherwise noted, all NMR spectrawere recorded in CDCl₃ at room temperature. ¹H chemical shifts arequoted in parts per million (δ in ppm) downfield from tetramethyl silane(TMS), which was used as an internal standard at 0 ppm and s, d, t, q, mdesignate singlet, doublet, triplet, quartet and multiplet,respectively. Melting points were determined using a Fisher-Johnsmelting apparatus and are uncorrected. UV spectra were recorded using aBeckmann Model # DU 7400 UV/V is spectrometer with a cell path length of1 cm. Methanol was used as the solvent for the UV spectra. FourierTransform Infrared spectra were recorded using an FT-Nicolet modelProtege #460 instrument. The infrared spectra of the liquid samples wererun as neat liquids using KBr discs. The KBr pellet method was used forall solid samples. The GC/mass spectrum analysis was conducted using aHewlett-Packard GC/mass spectrometer model #6890 equipped with a massion detector and Chem Station software. The temperature of the oven wassteadily increased from 70° C. to 250° C. and the carrier gas washelium.

TABLE 1 Organic Arsenic Acid Substituted Compounds No Ref. StructureFormula MW 1 P-273

C₂₂H₁₉AsN₅O₅ 508 2 P-370

C₁₇H₁₆AsN₅O₃S 445 3 P-371

C₁₇H₁₄AsN₇O₃ 439 4 P-374

C₁₅H₁₅AsN₈O₃ 430 5 P-378

C₁₆H₁₆AsN₃O₅ 405 6 P-379

C₁₆H₁₆AsN₃O₅ 405 7 P-380

C₁₁H₁₂AsN₃O₃S 341 8 P-381

C₁₁H₁₂AsN₃O₃S 341 9 P-385

C₁₁H₁₀AsN₅O₃ 335 10 P-386

C₁₁H₁₀AsN₅O₃ 335

4-[(6′,7′-Dimethoxyquinazoline-4′)-aminophenylazo]phenyl arsenic acid(WHI-P273)

The yield 71.20%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ8.83(s, 1H, 2-H),8,35(s, 1H, 5-H), 8.18-7.97 (m, 8H, Ph—H), 7.37(s, 1H, 8-H), 4.05(s, 3H,—OCH₃), 3.99(s, 3H, —OCH₃). UV(MeOH): 204.0, 215.0, 250.0, 330.0 nm.IR(KBr)υ_(max): 3431, 2629, 1675, 1580 cm⁻¹. Found: C,40.91; H,3.35; N,10.56. C₂₃H₂₂AsN₅O₅. 4HCl requires: C, 41.37; H, 3.90; N, 10.47%.

2-Methylthio-4-[(4′-aminophenylazo)-phenyl arsenicacid]pyrimidine(WHI-P370)

The yield 86.50%;. m.p. 240.0-242.0° C. ¹H NMR(DMSO-d₆): δ11.57(s, 1H,—NH), 8.23 (d, 1H, J=6.3 Hz, 5-H), 8.07-7.92 (m, 8H, Ph—H), 6.94(d, 1H,J=6.3 Hz, 6-H), 2.61(s, 3H, —SCH₃). UV(MeOH): 205.0, 215.0, 250.0, 330.0nm. IR(KBr)υ_(max): 3531, 2638, 1685, 1574 cm⁻¹.

6-[(4′-Aminophenylazo)-phenyl arsenic acid]-purine (WHI-P371)

The yield 81.30%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ11.61(s, 1H, —NH),9.56(s, broad, 3H, -9-NH, —As(OH)₂), 8.23 (d, 1H, J=6.3 Hz, 5-H),8.83(s, 1H, -2H), 8.77(s, 1H, -8H), 8.32-7.91 (m, 8H, Ph—H) 6.94(d, 1H,J=6.3 Hz, 6-H), 2.61(s, 3H, —SCH₃). UV(MeOH): 200.0, 213.0, 247.0, 328.0nm. IR(KBr)υ_(max): 3549, 2638, 1674, 1574 cm⁻¹.

2,6-Diamino-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine(WHI-P374)

The yield 79.50%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ12.04(s, 1H, —NH),8.01-7.70(m, 8H, Ph—H), 5.81(s, broad, 6H, —NH₂, —As(OH)₂). UV(MeOH):200.0, 213.0, 247.0, 328.0 nm. IR(KBr)υ_(max): 3400-3500, 2638, 1674,1574 cm⁻¹.

2,6-Dimethoxyl4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine(WHI-P376)

Yield 82.43%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ11.17(s, 1H, —NH),7.99-6.71(m, 8H, Ph—H), 3.86(s, 6H, —OCH₃). UV(MeOH): 203.0, 214.0, nm.IR(KBr)δ_(max): 3350-3550, 2640, 1633, cm⁻¹.

4-(2′-phenyl arsenic acid)amino-6,7-dimethoxyquinazoline (WHI-P378)

The yield 83.20%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ8.89(s, 1H, 2-H),8,21(s, 1H, 5-H), 8.46-7.51 (m, 4H, Ph—H), 7.40(s, 1H, 8-H), 3.99(s, 6H,—OCH₃). UV(MeOH): 208.0, 225.0, 253.0, 328.0 nn. IR(KBr)υ_(max): 3431,2629, 1685, 1580 cm³¹ ¹.

4-(4′-phenyl arsenic acid)-amino-6,7-dimethoxyquinazoline (WHI-P379)

The yield 85.40%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ11.33(s, 1H, —NH),8.23 (d, 1H, J=6.3 Hz, 5-H), 8.89(s, 1H, −2H), 8.26 (s, 1H, −8H),8.32-7.91 (m, 8H, Ph—H) 6.94(d, 1H, J=6.3 Hz, 6-H), 2.61(s, 3H, —SCH₃).UV(MeOH): 200.0, 213.0, 247.0, 328.0 nm. IR(KBr)υ_(max): 3549, 2638,1674, 1574 cm⁻¹.

2-Methylthio-4-(4′-phenyl arsenic acid)-aminopyrimidine (WHI-P380)

The yield 82.50%; m.p.>300.0° C. ¹H NMR(DMSO-d₆): δ11.19(s, 1H, —NH),9.82(s. broad, 2H, —As(OH)₂), 8.22 (d, 1H, J=6.3 Hz, 5-H), 7.98 (d, 2H,J=8.4 Hz, 2′,6′-H),),7.89 (d, 2H, J=8.4 Hz, 3,5′-H), 6.82 (d, 1H, J=6.3Hz, 6-H), 2.58(s, 3H, —SCH₃). UV(MeOH): 205.0, 213.0, 247.0, 328.0 nm.IR(KBr)υ_(max): 3400-3550, 2638, 1654, 1580 cm⁻¹.

2-Methylthio4-(2′-phenyl arsenic acid)-aminopyrimidine (WHI-P381)

The yield 86.40%; m.p. 225.0-228.0° C. ¹H NMR(DMSO-d₆): δ10.94(s, 1H,—NH), 8.46 (d, 1H, J=8.1 Hz, 5-H), 8.21-7.23(m, 4H, 3′, 4′, 5′, 6′-H),6.47(d, 1H, J=8.1 Hz, 6H), 2.49(s, 3H, —SCH₃). ¹³C NMR(DMSO-d₆):δ170.7(2-C), 159.0(4-C), 156.1(6-C), 141.7(5-C), 133.9(1′-C),131.5(6′-C), 123.3, 121.9, 121.0(3′, 4′, 5′-C), 103.6(2′-C),13.8(SCH₃-C). UV(MeOH): 201.0, 213.0, 247.0, 328.0 nm. IR(KBr)υ_(max):3420-3550, 2638, 1664, 1583 cm⁻¹.

6-(4′-phenyl arsenic acid)-aminopurine (WHI-P385)

The yield 71.30%; m.p.>300.0 ° C. ¹H NMR(DMSO-d₆): S 11.17(s, 1H, 6-NH),10.11(s, broad, 3H, 9-NH, As(OH)₂), 8.71 (s, 1H, 2-H), 8.67(s, 1H, 8-H),8.24 (d, 2H, J=8.7 Hz, 2′, 6′,-H), 7.79(d, 2H, J=8.7 Hz, 3′, 5′-H).UV(MeOH): 205.0, 213.0, 247.0, 328.0 nm. IR(KBr)υ_(max): 3350-3560,2638, 1678, 1582 cm⁻¹.

6-(2′-phenyl arsenic acid)-aminopurine (WHI-P386)

The yield 73.40%; m.p. 288.0-290.0° C. ¹H NMR(DMSO-d₆): δ11.34(s, 1H,6-NH), 8.75 (3,1H, 9-NH), 8.47(s,1H,2-H), 8.34(s, 1H, 8-H), 8.04-7.27(m,4H, 3′, 4′, 5′, 6′-H). UV(MeOH): 201.0, 213.0, 247.0, 328.0 nm.IR(KBr)υ_(max): 3430-3560, 2638, 1664, 1583 cm⁻¹.

Example 3 Cytotoxicity of Organic Arsenic Acid Substituted Compounds

The cytotoxicity of the organic arsenic acid substituted compoundsagainst specific tumor cells was evaluated using the MTT assay describedbelow.

Cytotoxicity Assay

The cytotoxicity assay of various compounds against tumor cells wasperformed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Boehringer Mannheim Corp., Indianapolis,Ind.). Unless otherwise specified, all cell lines were obtained from theAmerican Type Culture Collection (ATCC). Briefly, exponentially growingcells were seeded into a 96-well plate at a density of 2.5×10⁴cells/well and incubated for 36 hours at 37° C. prior to drug exposure.On the day of treatment, culture medium was carefully aspirated from thewells and replaced with fresh medium containing the organic arsenic acidsubstituted compounds WHI-P273, WHI-P370, WHI-P371, WHI-P374, WHI-P376,WHI-P378, WHI-P379, WHI-P380, WHI-P381, WHI-P385, or WHI-P386 atconcentrations ranging from 0.1 to 250 μM. Triplicate wells were usedfor each treatment.

Human leukemic cell lines (NALM-6, MOLT-3, ALL1, and RS4;11) and humanbreast tumor cell line (BT20) were obtained from the American TypeCulture Collection and maintained as a continuous cell line inDulbecco's modified Eagles's medium supplemented with 10% fetal bovineserum and antibiotics.

The cells were incubated with the various compounds for 24-36 hours at37° C. in a humidified 5% CO₂ atmosphere. To each well, 10 μl of MTT(0.5 mg/ml final concentration) was added and the plates were incubatedat 37° C. for 4 hours to allow MTT to form formazan crystals by reactingwith metabolically active cells. The formazan crystals were solubilizedovernight at 37° C. in a solution containing 10% SDS in 0.01 M HCl. Theabsorbence of each well was measured in a microplate reader (Labsystems)at 540 nm and a reference wavelength of 690 nm. To translate the OD₅₄₀values into the number of live cells in each well, the OD₅₄₀ values werecompared to those on standard OD₅₄₀-versus-cell number curves generatedfor each cell line. The percent survival was calculated using theformula:${\% \quad {Survival}} = {\frac{{live}\quad {cell}\quad {{number}\quad\lbrack{test}\rbrack}}{{live}\quad {cell}\quad {{number}\quad\lbrack{control}\rbrack}} \times 100}$

The IC₅₀ values for cytotoxic activity were calculated by non-linearregression analysis, and are shown below in Table 2.

TABLE 2 Cytotoxic Activity of Organic arsenic Acid Substituted Compoundsagainst leukemic (NALM-6, MOLT-3, ALL1, RS4;11) and breast cancer (B120)cells. NALM-6 MOLT-3 ALL1 RS4;11 BT20 Drug IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)IC₅₀ (μM) IC₅₀ (μM) WHI-P273 7.3 12.1 8.2 7.7 >250 WHI-P370 5.98 2.9 1.94.8 67.8 WHI-P371 15.2 21.6 24.5 16.2 >250 WHI-P374 50.1 103.6 55.8 53.613.9 WHI-P376 64.8 50.4 29.1 23.3 21.5 WHI-P378 46.6 67.3 128.3114.7 >250 WHI-P380 1.7 14.2 6.2 10.2 238.6 WHI-P381 <1.9 <1.9 <1.9 <1.917.3 WHI-P386 30.8 >250 >250 >250 >250

Each of the organic arsenic substituted compounds used in this studyexhibited a potent cytotoxic effect against at least one of the tumorcells used. Compounds, WHI-P370, WHI-P374, WHI-P376 and WHI-P381 werecytotoxic against the breast cancer cell line, BT20, while compoundsWHI-P273, WHI-P370, WHI-P371, WHI-P376, WHI-P380 and WHI-P381 werecytotoxic for all of the leukemic cell lines used. As shown in Table 2,WHI-P380 and WHI-P381 exhibited highest cytotoxic activity, causing celldeath in leukemic cell lines at micromolar concentrations with IC₅₀values of 1.7 to 14.2 μM (WHI-P380) and 1.9 μm (WHI-P281)

The dose-responsive anti-proliferative activity of various organicarsenic acid substituted compounds against leukemic cell lines (NALM-6and MOLT-3) is shown below in Table 3. FIGS. 3A-3F show dose responsecurves in ALL1, NALM-6, and MOLT-3 cells.

TABLE 3 Anti-proliferative Activity of Organic Arsenic SubstitutedCompounds Against Leukemic Cell Lines. Concentration Mean No. of % DrugCell Line (mM) Colonies/10⁶ Cells Inhibition WHI-P273 NALM-6 04123(4068, 4364) — 0.1 3638(3260, 4016) 13.7 1 61(24, 96) 98.5 10 0 100100 0 100 MOLT-3 0 1056(1000, 1112) — 0.1 1230(968, 1492) — 1 264(216,312) 75 10 0 100 100 0 100 WHI-P370 NALM-6 0 4216(4068, 4364) — 0.12194(2860, 2968) 30.9 1 0 100 10 0 100 100 0 100 MOLT-3 0 1056(1000,1112) — 0.1 992(896, 1088) 6.1 1 0 100 10 0 100 100 0 100 WHI-P371NALM-6 0 4216(4068, 4364) — 0.1 3846(3696, 3996) 8.8 1 994(616, 1372)76.4 10 0 100 100 0 100 MOLT-3 0 1056(1000, 1112) — 0.1 1098(1004, 1192)— 1 761(678, 844) 27.9 10 0 100 100 0 100 WHI-P380 NALM-6 0 1208(864,1552) — 0.1 400(116, 684) 66.9 1 2(3, 1) 99.8 10 0 100 100 0 100 MOLT-30 1438(1424, 1452) — 0.1 678(624, 732) 52.8 1 436(272, 600) 69.7 10 0100 100 0 100 WHI-P381 NALM-6 0 1208(864, 1152) — 0.1 0 100 1 0 100 10 0100 100 0 100 MOLT-3 0 1438(1424, 1452) — 0.1 429(202, 656) 70.2 1 0 10010 0 100 100 0 100

Example 4 Organic Arsenic Acid Substituted Compounds Induce Apoptosis inCancer Cells

In situ Detection of Apoptosis

Assay for apoptosis was performed by the in situ nick-end-labelingmethod using an ApopTag in situ detection kit (Oncor, Gaithersburg, Md.)according to the manufacturer's recommendations. Exponentially growingcells (NALM-6 and MOLT-3) were seeded in 6-well tissue culture plates ata density of 50×10⁴ cells/well and cultured for 36 hours at 37° C. in ahumidified 5% CO₂ atmosphere. The supernatant culture medium wascarefully aspirated and replaced with fresh medium alone or fresh mediumcontaining WHI-P381 at a concentration of 2 μg/ml.

After a 36 hour incubation at 37° C. in a humidified 5% CO₂ incubator,the supernatants were carefully aspirated and the cells were treated for1-2 minutes with 0.1% trypsin. The detached cells were collected into a15 ml centrifuge tube, washed with medium and pelleted by centrifugationat 1000 rpm for 5 minutes. Cells were resuspended in 50 μl of PBS,transferred to poly-L-lysine coated coverslips and allowed to attach for15 minutes. The cells were washed once with PBS and incubated withequilibration buffer for 10 minutes at room temperature.

After removal of the equilibration buffer, cells were incubated for 1hour at 37° C. with the reaction mixture containing terminaldeoxynucleotidyl transferase (TdT) and digoxigenin-11-UTP for labelingof exposed 3′-hydroxyl ends of fragmented nuclear DNA. The cells werewashed with PBS and incubated with anti-digoxigenin antibody conjugatedto FITC for 1 hour at room temperature to detect the incorporated dUTP.After washing the cells with PBS, the coverslips were mounted ontoslides with Vectashield containing propidium iodide (Vector Labs,Burlingame, Calif.) and viewed with a confocal laser scanningmicroscope. Non-apoptotic cells do not incorporate significant amountsof dUTP due to lack of exposed 3-hydroxyl ends, and consequently havemuch less fluorescence than apoptotic cells, which have an abundance ofexposed 3′-hydroxyl ends. In control reactions, the TdT enzyme wasomitted from the reaction mixture.

Results

The ability of the organic arsenic substituted compound, WHI-P381, toinduce apoptotic cell death in leukemic cell lines, NALM-6 and MOLT-3,is shown in FIG. 1. Treated cells, NALM-6 (FIG. 1B) and MOLT-3 (FIG. 1D)showed a much greater Fluorescence, percentage of apoptosis, thanuntreated cells, NALM-6 (FIG. 1A) and MOLT-3 (FIG. 1C). These resultsdemonstrate the apoptosis-inducing activity of the compounds of theinvention.

Example 5 Organic Arsenic Compounds are More Potent Than ArsenicTrioxide

Leukemic NALM-6 cells were treated with WHI-P381 or arsenic trioxide atdoses from 0.07 μM to 5 μM for 1, 2, 3 or 4 days. Cell survival wasassessed by the MTT assay and IC₅₀ calculated as described above.Surprisingly, as shown in FIGS. 2A-2D, the organic arsenic acidsubstituted compound, WHI-P381 was more cytotoxic than arsenic trioxide,which has been shown to induce apoptosis in refractory acute promyelicleukemia cells, B-cell leukemic cells and megakaryocytotic leukemia cellline. The time and dose-dependent activity of WHI-P381 in comparisonwith arsenic trioxide is shown in FIGS. 2A-2D. The IC₅₀ values forWHI-P381 are shown in Table 4 below, and demonstrate the more potentactivity of the organic arsenic compound of the invention over arsenictrioxide.

TABLE 4 Cytotoxicity against NALM-6 cells. IC₅₀ (μM) Day 1 Day 2 Day 3Day 4 WHI-P381 2.03 0.85 0.7 0.68 Arsenic trioxide 3.8 3.2 2.0 1.2

Example 6 Organic Arsenic Acid Substituted Compounds are Cytotoxic toPrimary Leukemic Cells

The cytotoxic activity of a variety of organic arsenic acid substitutedcompounds against primary leukemic cells was determined using MTT assaysas described above. Cells obtained from 5 leukemia patients were treatedwith WHI-P273, WHI-P370, WHI-P371, WHI-P380 and WHI-P381 according tothe methods described above for cell lines. Cytotoxicity was assessed bythe methods described above. The data are presented in lo FIGS. 4A-4E,and demonstrate that primary leukemic cells are susceptible to thecytotoxic effects of the organic arsenic acid substituted compounds.

All publications, patents, and patent documents described herein areincorporated by reference as if fully set forth. The invention describedherein may be modified to include alternative embodiments. All suchobvious alternatives are within the spirit and scope of the invention,as claimed below.

We claim:
 1. A compound of the formula

R¹ is H, NR³R⁴, SR³, OR³, in which R³ and R⁴ are each independentlyhydrogen or a C₁-C₄ alkyl group; X is N; R² is H, NR³R⁴, SR³, OR³, inwhich R³ and R⁴ are each independently hydrogen or a C₁-C₄ alkyl group;or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, wherein R³ is hydrogen or methyl, and R⁴ is hydrogen.
 3. The compoundof claim 1, wherein the compound is2,6-Diamino-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine.4. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of claim 1 and a pharmaceutically acceptablediluent or carrier.
 5. A method for inhibiting the growth of tumor cellsin a subject comprising administering to said subject on effectiveamount of a compound of claim
 1. 6. The method of claim 5, wherein saidinhibiting comprises inducing apoptosis in said tumor cells.
 7. A methodof treating cancer selected from, the group consisting of leukemia andbreast cancer in a subject comprising administering to said subject atherapeutically effective amount of a compound of claim
 1. 8. The methodof claim 7, wherein said cancer is leukemia.
 9. The method of claim 7,wherein said cancer is breast cancer.
 10. A method for inducingcytotoxicity in a cell, comprising administering to said cell acytotoxic dose of the compound of claim
 1. 11. The method of claim 10,wherein said cell is a tumor cell.
 12. The method of claim 11, whereinsaid compound is 2,6-Diamino-4[(4′-aminophenylazo)-phenyl arsenicacid]-1,3,5-triazine, or 2,6-Dimethoxyl-4[(4′-aminophenylazo)-phenylarsenic acid]-1,3,5-triazine.
 13. The method of claim 12, wherein saidcompound is 2,6-Diamino-4[(4′-aminophenylazo)-phenyl arsenicacid]-1,3,5-triazine.
 14. A compound of the formula

wherein R is

R¹ is H, SR³, OR³, in which R³ is hydrogen or a C₁-C₄ alkyl group; X isN; R² is H, SR³, OR³, in which R³ is hydrogen or a C₁-C₄ alkyl group; ora pharmaceutically acceptable salt thereof.
 15. The compound of claim14, wherein the compound is 2,6-dimethoxy-4[(4′-aminophenylazo)-phenylarsenic acid]-1,3,5-triazine.
 16. A pharmaceutical compositioncomprising a therapeutically effective amount of a compound of claim 14and a pharmaceutically acceptable diluent or carrier.
 17. Thepharmaceutical composition of claim 14, wherein the compound is2,6-dimethoxy-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine.18. A method for inhibiting the growth of tumor cells in a subjectcomprising administering to said subject on effective amount of acompound of claim
 14. 19. The method of claim 18, wherein the compoundis 2,6-dimethoxy-4[(4′-aminophenylazo)-phenyl arsenicacid]-1,3,5-triazine.
 20. The pharmaceutical composition of claim 4,wherein the compound is 2,6-diamino-4[(4′-aminophenylazo)-phenyl arsenicacid]-1,3,5-triazine.
 21. The method of claim 5, wherein the compound is2,6-diamino-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine or2,6-dimethoxy-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine.22. The method of claim 9, wherein the compound is2,6-diamino-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine or2,6-dimethoxy-4[(4′-aminophenylazo)-phenyl arsenic acid]-1,3,5-triazine.